London Protocol under water-perfused HRM in a healthy population, towards novel 3D manometric parameters in an evaluation of anorectal functional disorders.

BACKGROUND/AIM: London Protocol (LP) and Classification allied to high-resolution manometry (HRM) technological evolution has updated and enhanced the diagnostic armamentarium in anorectal disorders. This study aims to evaluate LP reproducibility under water-perfused HRM, provide normal data and new parameters based on 3D and healthy comparison studies under perfusional HRM. METHODS: Fifty healthy (25 F) underwent water-perfused 36 channel HRM based on LP at resting, squeeze, cough, push, and rectal sensory. Additional 3D manometric parameters were: pressure-volume (PV) 104mmHg2.cm (resting, short and long squeeze, cough); highest and lowest pressure asymmetry (resting, short squeeze, and cough). Complementary parameters (CP) were: resting (mean pressure, functional anal canal length); short squeeze (mean and maximum absolute squeeze pressure), endurance (fatigue rate, fatigue rate index, capacity to sustain); cough (anorectal gradient pressure); push (rectum-anal gradient pressure, anal canal relaxation percent); recto-anal inhibitory reflex (anal canal relaxation percent). RESULTS: No difference to genders: resting (LP, CP, and 3D); short squeeze (highest pressure asymmetry); endurance (CP); cough (CP, highest and lowest pressure asymmetry); push (gradient pressure); rectal sensory. Higher pressure in men: short squeeze (maximum incremental, absolute, and mean pressure, PV, lowest pressure asymmetry); long squeeze (PV); cough (anal canal and rectum maximum pressure, anal canal PV); push (anal canal and rectum maximum pressure). Anal canal relaxation was higher in women (push). CONCLUSIONS: LP reproducibility is feasible under water-perfused HRM, and comparative studies could bring similarity to dataset expansion. Novel 3D parameters need further studies with healthy and larger data to be validated and for disease comparisons. KEY POINTS: • London Protocol and Classification allied with the technological evolution of HRM (software and probes) has refined the diagnostic armamentarium in anorectal disorders. • Novel 3D and deepening the analysis of manometric parameters before the London Classification as a contributory diagnostic tool. • Comparison of healthy volunteers according to the London Protocol under a perfusional high-resolution system could establish equivalence points.

STOPP-START Criteria Used to Identify the Elderly Population Prone to Potentially Inadequate Prescribing in a Colombian Cohort.

BACKGROUND: Elderly people have multiple comorbidities that often require treatment with multiple medications. Having strategies to lessen the risks associated with pharmacological interactions and potentially inadequate prescribing (PIP) is of major importance. The STOPP- START criteria are useful in identifying PIP along with other tools, such as LASA (look alike/sound alike) drugs and high-risk medications (HRM). OBJECTIVE: We aimed to clinically and sociodemographically characterize the population with PIP according to the STOPP-START criteria in hospitalized elderly patients over 6 months in a third-level hospital in Colombia, South America. We also aimed to calculate the prevalence of PIP, LASA drugs and HRM and to identify other problems related with medication. Finally, we proposed an algorithm for the identification of PIP in this population. METHODS AND MATERIALS: This was a descriptive, cross-sectional study in hospitalized patients older than 60 years during the first semester of 2021 to identify PIP according to STOPP- START criteria. An analysis of clinical and sociodemographic variables was conducted, as well as the construction of an algorithm to identify PIP in the elderly in a semiautomated way. Data were collected and analyzed using the software SPSS 2021, using descriptive statistics and measures of central tendency. RESULTS: The prevalence of PIP in the study population was 25%. Furthermore, 60% of patients had one problem related to medication, and 27% used at least one LASA drug or HRM. CONCLUSION: This study allows one to characterize, for the first time, the Colombian population prone to PIP, as well as the construction of an algorithm that identifies PIP in a semiautomated way.

HRM Accuracy and Limitations as a Species Typing Tool for Leishmania Parasites.

High Resolution Melting Analysis (HRM) has been pointed out as a suitable alternative method to detect and identify Leishmania species. Herein, we aimed to evaluate the sensitivity, specificity, accuracy, and limitations of a HSP70-HRM protocol both as a diagnostic scheme applied in clinical samples and as a species typing tool for laboratory research and reference services. Our data reveal the pronounced species-typing potential of the HSP70-HRM in DNA from cultured parasites. For clinical samples, however, we advise caution due to parasite load-dependent accuracy. In light of these findings and considering the importance of parasite load determination for clinical and research purposes, we recommend the integration of the presented typing scheme and the previously published Leishmania quantifying approach as combined tools for clinicians, surveillance, and research.

Detection of Leishmania spp. in Cats: Analysis of Nasal, Oral and Conjunctival Swabs by PCR and HRM.

BACKGROUND AND OBJECTIVES: Feline leishmaniasis (FeL) is caused by several species of parasites of the genus Leishmania. The disease can occur with the presence or absence of clinical signs, similar to those observed in other common infectious diseases. In endemic regions for FeL, the infection has been associated with dermatological lesions. Therefore, considering the search for less invasive and more effective diagnostic techniques, we aimed to investigate the presence of Leishmania spp. in domestic cats through Polymerase Chain Reaction (PCR) and high-resolution melting (HRM) analyses of conjunctival, oral, and nasal epithelial cells, and we detected the presence of anti-Leishmania IgG antibodies from serological techniques of the Immunofluorescent Antibody Test (IFAT) and ELISA. METHODS: The PCR and HRM for detection of Leishmania spp. were performed on 36 samples of epithelial cells from the conjunctiva of male and female cats, collected using sterile swabs. The serological tests IFAT and ELISA were also performed. RESULTS: The prevalence of Leishmania donovani infection was 11.1% (4/36) by PCR assay, and those results were confirmed for Leishmania species using the HRM technique. Twenty-four cats (24/36 = 66.7%) were reactive to the IFAT and twenty-two cats were reactive by the ELISA technique (22/36 = 61.1%). INTERPRETATION AND CONCLUSIONS: The use of conjunctival swabs was shown to be a non-invasive, practical, and easy-to-perform technique, and in addition to the genetic sequencing and HRM, it was able to identify the parasitic DNA of L. donovani in cats. This technique can be used for screening diagnosis in future epidemiological surveys of FeL and can be used as a complement to clinical and/or serological tests, as well as associating the clinical history of the animal, for the diagnostic conclusion.

Potential use of high-resolution melting analyses for SARS-CoV-2 genomic surveillance.

The pandemic caused by COVID-19 and the emergence of new variants of SARS-CoV-2 have generated clinical and epidemiological impacts on a global scale. The use of strategies for monitoring viral circulation and identifying mutations in genomic regions involved in host interaction are important measures to mitigate viral dissemination and reduce its likely complications on population health. In this context, the objective of this work was to explore the potential of high-resolution melting (HRM) analysis combined with one-step real-time reverse transcription PCR in a closed-tube system, as a fast and convenient method of screening for SARS-CoV-2 mutations with possible implications on host-pathogen interactions. The HRM analyses allowed the distinction of the Gamma, Zeta, Alpha, Delta, and Omicron variants against the predecessors (B.1.1.28, B.1.1.33) of occurrence in Brazil. It is concluded that the molecular tool standardized here has the potential to optimize the genomic surveillance of SARS-CoV-2, and could be adapted for genomic surveillance of other pathogens, due to its ability to detect, prior to sequencing, samples suggestive of new variants, selecting them more assertively and earlier for whole genome sequencing when compared to random screening.

Exploring the Evolution of Human Resource Analytics: A Bibliometric Study.

The objective of this study is to identify and analyze the most relevant scientific work being undertaken in HR analytics. Additionally, it is to understand the evolution of the conceptual, intellectual, and social structure of this topic in a way that allows the expansion of empirical and conceptual knowledge. Bibliometric analysis was performed using Bibliometrix and Biblioshiny software packages on academic articles indexed on the Scopus and Web of Science (WoS) databases. Search criteria were applied, initially resulting in a total of 331 articles in the period 2008-2022. Finally, after applying exclusion criteria, a total of 218 articles of interest were obtained. The results of this research present the relevant notable topics in HR analytics, providing a quantitative analysis that gives an overview of HR analytics featuring tables, graphs, and maps, as well as identifying the main performance indicators for the production of articles and their citations. The scientific literature on HR analytics is a novel, adaptive area that provides the option to transform traditional HR practices. Through the use of technology, HR analytics can improve HR strategies and organisational performance, as well as people's experiences.

High prevalence of intermediate resistance to ciprofloxacin in Salmonella enterica isolated from a Brazilian poultry production chain, located in Minas Gerais state.

We characterized the distribution and diversity of antimicrobial-resistance Salmonella enterica isolated from a poultry production chain in Minas Gerais, Brazil, with special attention to ciprofloxacin and multidrug resistance (MDR). S. enterica (n = 96) of different serotypes and from different processing steps were subjected to broth dilution assay to estimate the minimum inhibitory concentration (MIC) for 12 antibiotics (8 classes) and screened using PCR for the presence of 17 antimicrobial-resistance genes. Isolates presented mainly resistance to ampicillin (11/96), and most presented intermediate resistance to ciprofloxacin (92/96). Roughly one-third (33/96) were resistant to streptomycin based on our interpretive criteria. Most strains resistant to streptomycin and ciprofloxacin were PCR-positive for aphA (51/96) and qnrB (94/96), respectively. Ciprofloxacin resistance was further investigated through high-resolution melting qPCR (HRM-qPCR) and sequencing of quinolone resistance-determining region (QRDR: gyrA, gyrB, parC, and parE). Minor differences were identified in melting temperatures (Tm), and a Thr57Sr mutation was observed in parC. MDR isolates harboring acrA and capable of expressing the AcrAB-TolC multidrug efflux pump were resistant to ethidium bromide at 0.4 mg/mL. The intermediate resistance to ciprofloxacin may be associated with qnrB, and the potential role of Thr57Ser mutation warrants further investigation. The high prevalence of antibiotic related genes and its association with the observed intermediary resistance to ciprofloxacin indicates the widespread of this hazard in the studied poultry production chain.

Accurate, fast and cost-effective simultaneous detection of bacterial meningitis by qualitative PCR with high-resolution melting.

Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae are leading causes of meningitis and acute invasive infections. PCR-based methods are widely used for the diagnosis and surveillance of bacterial pathogens because of their high sensitivity, specificity and high-throughput capabilities compared with conventional laboratory methods. This study evaluated a high-resolution melting qualitative PCR analysis method for the simultaneous detection of these three pathogens. The assay has been optimized to detect three species-specific genes of each organism isolated from clinical samples, enabling accurate identification of the etiological agent. The method proved to be highly sensitive and cheaper than the real-time PCR TaqMan® system because it is probe-free; it could be used for the diagnosis of invasive diseases in public health laboratories of developing countries.

Application of MaxEnt Modeling and HRM Analysis to Support the Conservation and Domestication of Gevuina avellana Mol. in Central Chile.

The Chilean hazelnut (Gevuina avellana Mol., Proteaceae) is a native tree of Chile and Argentina of edible fruit-type nut. We applied two approaches to contribute to the development of strategies for mitigation of the effects of climate change and anthropic activities in G. avellana. It corresponds to the first report where both tools are integrated, the MaxEnt model to predict the current and future potential distribution coupled with High-Resolution Melting Analysis (HRM) to assess its genetic diversity and understand how the species would respond to these changes. Two global climate models: CNRM-CM6-1 and MIROC-ES2L for four Shared Socioeconomic Pathways: 126, 245, 370, and 585 (2021−2040; 2061−2080) were evaluated. The annual mean temperature (43.7%) and water steam (23.4%) were the key factors for the distribution current of G. avellana (AUC = 0.953). The future prediction model shows to the year 2040 those habitat range decreases at 50% (AUC = 0.918). The genetic structure was investigated in seven natural populations using eight EST-SSR markers, showing a percentage of polymorphic loci between 18.69 and 55.14% and low genetic differentiation between populations (Fst = 0.052; p < 0.001). According to the discriminant analysis of principal components (DAPC) we identified 10 genetic populations. We conclude that high-priority areas for protection correspond to Los Avellanos and Punta de Águila populations due to their greater genetic diversity and allelic richness.

Comparison of two commercial methods for smooth-shelled mussels (Mytilus spp.) species identification.

Seafood international trade has increased the labeling requirements in standards and regulations to include product information that enable traders and consumers to make informed choices. The European Union (EU) Regulation No. 1379/2013 imposes the declaration of an official commercial designation and scientific names for all the fishery and aquaculture products to be offered for sale to the final consumers. DNA analyses are used to enforce this regulation and to test authenticity in processed foods. We compared the performance of two mono-locus approaches for species identification (SI) in 61 Mytilus mussels: the high-resolution melting analysis of the polyphenolic adhesive protein gene and the partial sequencing of the histone H1C gene. The H1C sequences were analyzed with five different methods. Both approaches show discrepancies in the identification of putative hybrids (0.0 < κ < 0.687 and 0.0 < MCC < 0.724). Excluding putative hybrids, methods show substantial to perfect agreement (0.772 < κ < 1.0 and 0.783 < MCC < 1.0). This study highlights the need to use standardized molecular tools, as well as to use multi-locus methods for SI of Mytilus mussels in testing laboratories.

A sensitive and inexpensive high-resolution melting-based testing algorithm for diagnosis of transient abnormal myelopoiesis and myeloid leukemia of Down syndrome.

Patients with Down syndrome (DS) are commonly affected by a pre-leukemic disorder known as transient abnormal myelopoiesis (TAM). This condition usually undergoes spontaneous remission within the first 2 months after birth; however, in children under 5, 20%-30% of cases evolve to myeloid leukemia of Down syndrome (ML-DS). TAM and ML-DS are caused by co-operation between trisomy 21 and acquired mutations in the GATA1 gene. Currently, only next-generation sequencing (NGS)-based methodologies are sufficiently sensitive for diagnosis in samples with small GATA1 mutant clones (≤10% blasts). Alternatively, this study presents research on a new, fast, sensitive, and inexpensive high-resolution melting (HRM)-based diagnostic approach that allows the detection of most cases of GATA1 mutations, including silent TAM. The algorithm first uses flow cytometry for blast count, followed by HRM and Sanger sequencing to search for mutations on exons 2 and 3 of GATA1. We analyzed 138 samples of DS patients: 110 of asymptomatic neonates, 10 suspected of having TAM, and 18 suspected of having ML-DS. Our algorithm enabled the identification of 33 mutant samples, among them five cases of silent TAM (5/110) and seven cases of ML-DS (7/18) with blast count ≤10%, in which GATA1 alterations were easily detected by HRM. Depending on the type of genetic variation and its location, our methodology reached sensitivity similar to that obtained by NGS (0.3%) at a considerably reduced time and cost, thus making it accessible worldwide.

Molecular characterization of Burkholderia mallei strains isolated from horses in Brazil (2014-2017).

Glanders is an infectious zoonosis caused by Burkholderia (B.) mallei that mainly affects equids. The objective of this work was to provide additional knowledge on the diversity of the strains circulating in Brazil. Six Burkholderia mallei isolates obtained during necropsies of glanderous horses between 2014 and 2017 in two different states (Pernambuco and Alagoas) were analyzed by polymerase chain reaction-high-resolution melting (PCR-HRM). While four strains (9902 RSC, BM_campo 1, BM_campo 3 and UFAL2) clustered in the L3B2 branch, which already includes the Brazilian 16-2438_BM#8 strain, two strains (BM_campo 2.1 and BM_campo 2.2) clustered within the L3B3sB3 branch, which mostly includes older isolates, from Europe and the Middle East. Whole genome sequencing of two of these strains (UFAL2 and BM_campo 2.1), belonging to different branches, confirmed the HRM typing results and refined the links between the strains, including the description of the L3B3Sb3Gp1SbGp1 genotype, never reported so far for contemporary strains. These results suggest different glanders introduction events in Brazil, including a potential link with strains of European origin, related to colonization or trade.

Cannabis Seeds Authentication by Chloroplast and Nuclear DNA Analysis Coupled with High-Resolution Melting Method for Quality Control Purposes.

Background: Cannabis plants and their seed have been used in many cultures as a source of medicine and feeding during history. Today, there is an increasing demand for cannabis seeds for medical use. Moreover, a seed sales market with no legal regulations has also grown. This may pose some issues if a quality control is not set in place. Identification of cannabis strains is important for quality control purposes in a nonregulated growing market and in cases of illegal traffic and medical use. Owing to the high price as a pharmacological drug, commercial products of cannabis plants and seeds for medical users are often subjected to adulterations, either when packing or distributing certified seeds in the market. Materials and Methods: Cannabis commercial seeds and cannabis seeds for medical use were analyzed with high-resolution melting (HRM) analysis using barcoding markers. Humulus lupulus L. plants from a local market were used as outgroup control. DNA barcoding uses specific regions of the genome to identify differences in the genetic sequence of conserved regions such as internal transcribed spacer (ITS) and rbcL. DNA barcoding data can be generated with real-time polymerase chain reaction combined with HRM analysis to distinguish specific conserved DNA regions of closely related species. HRM analysis is the method of choice for rapid analysis of sequence variation. Results: The melting temperature (Tm) of homogeneous packages was consistent with single genotypes. However, packages containing contaminating seeds showed Tm differences of 0.2°C on average. Conclusions: An effective, rapid, and low-cost method based on ITS nuclear DNA and on chloroplast rbcL regions for screening and detection of contamination in commercial cannabis seeds was developed and applied for the analysis of different samples. This approach can be used as a quality control tool for cannabis seeds or other plant material.

Sensitivity comparison for the Leishmania spp. detection in different canine tissues using PCR-HRM

ABSTRACT Background: Leishmaniasis is a vector-borne disease caused by a parasite protozoon from the genus Leishmania. Among the molecular techniques applied for detecting these parasites, real-time PCR with High Resolution Melting (PCR-HRM) proved advantageous since it simultaneously determines both the presence and species of the pathogen in one step, through amplification and later analysis of curves generated by melting temperature. Methods: Based on this molecular technique, the goal of this study was to estimate the PCR-HRM sensitivity for Leishmania spp. detection in different canine tissues by evaluating biological samples obtained from popliteal, submandibular, and pre-scapular lymph nodes, from bone marrow and ear pinnae of 28 stray dogs captured in the metropolitan area of Asunción (Paraguay). Results: The rk39 immunochromatographic test showed that 25/28 tested dogs (89%) presented antibodies against L. infantum. In 20/25 dogs that tested positive for rk39 (80%), it was possible to detect Leishmania spp. by PCR-HRM and determine that the species corresponded entirely to L. infantum. Regarding the analysis of different tissues, the parasite was detected in all popliteal lymph node samples, followed by high detection in submandibular (at 95%) and pre-scapular lymph nodes (at 90%), bone marrow (at 85%), and ear pinnae (at 85%). Conclusions: This study demonstrated that the use of real-time PCR-HRM using the molecular marker hsp70 was a highly sensitive method for simultaneously detecting and identifying Leishmania species in different tissues taken from infected dogs. In addition, the usefulness of ear pinnae as easily accessible tissue for molecular diagnosis was emphasized.

Two Leishmania species separation targeting the ITS-rDNA and Cyt b genes by developing and evaluating HRM- qPCR

ABSTRACT Background: Incidence of Cutaneous Leishmaniasis as an infectious and neglected disease is increasing, for the diagnosis of which several traditional methods and conventional PCR techniques have been developed, employing different genes for species identification. Methods: Leishmania parasites were sampled, DNA was extracted, and new specific and sensitive primers were designed. Two ITS-rDNA and Cyt b genes were targeted by qPCR using the High- Resolution Melting method to identify Leishmania parasites. The standard curves were drawn, compared, and identified by high-resolution melting curve analysis. Results: Melting temperature and Cycle of Threshold of ITS-rDNA was higher than Cyt b but Cyt b was more sensitive than ITS-rDNA when Leishmania major and Leishmania tropica were analyzed and evaluated. By aligning melt curves, normalizing fluorescence curves, and difference plotting melt curves, each Leishmania species was distinguished easily. L. major and L. tropica were separated at 83.6 °C and 84.7 °C, respectively, with less than 0.9 °C of temperature difference. Developing sensitivity and specificity of real-time PCR based on EvaGreen could detect DNA concentration to less than one pmol. Conclusions: Precise identification of Leishmania parasites is crucial for strategies of disease control. Real-time PCR using EvaGreen provides rapid, highly sensitive, and specific detection of parasite's DNA. The modified High-Resolution Melting could determine unique curves and was able to detect single nucleotide polymorphisms according to small differences in the nucleotide content of Leishmania parasites.

Identificación de especies de Leishmania mediante PCR en tiempo real acoplada a curvas de fusión de alta resolución

RESUMEN Introducción: La leishmaniasis es una enfermedad causada por parásitos del género Leishmania. En Colombia se han informado 10 especies patógenas. El diagnóstico parasitológico tradicional basado en la observación de los parásitos no permite identificar la especie, por lo cual se deben emplear métodos moleculares, entre ellos la reacción en cadena de la polimerasa o PCR convencional, pero esta presenta algunas limitaciones y requiere extensos periodos de tiempo para la obtención de resultados, que en ocasiones no son concluyentes. Objetivo: Evaluar un método basado en PCR en tiempo real acoplado a curva de temperatura de desnaturalización media de alta resolución (PCR-HRM) que permita el diagnóstico y la identificación simultánea de parásitos del género Leishmania en muestras clínicas de humanos y en cultivos in vitro de manera sensible y específica. Métodos: Se estandarizó una PCR-HRM, mediante la cual se evaluaron 237 muestras clínicas, 98 clasificadas como positivas y 139 como negativas, parasitológicamente por directo y/o cultivo. Las tipificaciones fueron comparadas con los resultados en paralelo obtenidos de una variante de la PCR, realizando cortes al amplicon que generó un fragmento de restricción de longitud polimórfica o PCR-RFLP que había sido previamente estandarizada. Resultados: Se logró implementar una PCR-HRM para el diagnóstico e identificación de especies de Leishmania, logrando un 100 % de concordancia con las tipificaciones obtenidas por PCR-RFLP. Incluso, se logró detectar e identificar el parásito en muestras diagnosticadas como negativas por los métodos convencionales. Se encontró que con un porcentaje de confiabilidad superior al 95 %, se lograron tipificar 91 muestras de 98; de estas el 81,63 % de los casos fueron L. panamensis, el 11,22% L. braziliensis e indeterminadas el 7,14 % de los casos. Conclusiones: La PCR-HRM es un buen método que permite la identificación de las especies más prevalentes en Colombia, comparando temperaturas medias de desnaturalización específicas según la especie de Leishmania involucrada.

ABSTRACT Introduction: Leishmaniasis is a disease caused by parasites of the genus Leishmania. Ten pathogenic species have been reported in Colombia. Traditional parasite diagnosis based on observation of the parasites does not make it possible to identify the species. Therefore, it is necessary to use molecular methods, among them conventional polymerase chain reaction or PCR, but this test presents some limitations and requires long periods of time to obtain results which sometimes are not conclusive. Objective: Evaluate a method based on real time PCR coupled with high resolution mean denaturalization temperature curve analysis (HRM-PCR) for the diagnosis and simultaneous identification of parasites of the genus Leishmania in clinical samples from humans and in vitro cultures in a sensitive and specific manner. Methods: Standardization was performed of an HRM-PCR with which 237 clinical samples were evaluated, 98 classified as positive and 139 as negative, by direct parasitological examination and/or culture. The typing obtained was compared with parallel results from a PCR variant, making cuts on the amplicon that generated a restriction fragment length polymorphism or PCR-RFLP previously standardized. Results: An HRM-PCR could be implemented for the diagnosis and identification of Leishmania species, achieving 100% concordance with the typing obtained by PCR-RFLP. It was even possible to detect and identify the parasite in samples diagnosed as negative by conventional methods. Of the total 98 samples, 91 could be typed with a percentage of reliability above 95%. Of these, 81.63% of the cases were L. panamensis, 11.22% were L. braziliensis and 7.14 % were indeterminate. Conclusions: HRM-PCR is a good method to identify the species most prevalent in Colombia, comparing specific mean denaturalization temperatures according to the Leishmania species involved.

Genetic and Bioinformatic Strategies to Improve Diagnosis in Three Inherited Bleeding Disorders in Bogotá, Colombia.

Inherited bleeding disorders (IBDs) are the most frequent congenital diseases in the Colombian population; three of them are hemophilia A (HA), hemophilia B (HB), and von Willebrand Disease (VWD). Currently, diagnosis relies on multiple clinical laboratory assays to assign a phenotype. Due to the lack of accessibility to these tests, patients can receive an incomplete diagnosis. In these cases, genetic studies reinforce the clinical diagnosis. The present study characterized the molecular genetic basis of 11 HA, three HB, and five VWD patients by sequencing the F8, F9, or the VWF gene. Twelve variations were found in HA patients, four in HB patients, and 19 in WVD patients. From these variations a total of 25 novel variations were found. Disease-causing variations were used as positive controls for validation of the high-resolution melting (HRM) variant-scanning technique. This approach is a low-cost genetic diagnostic method proposed to be incorporated in developing countries. For the data analysis, we developed an accessible open-source code in Python that improves HRM data analysis with better sensitivity of 95% and without bias when using different HRM equipment and software. Analysis of amplicons with a length greater than 300 bp can be performed by implementing an analysis by denaturation domains.

Evaluation of filter paper to transport inactivated bacteria to detect carbapenem resistance genes by multiplex real-time PCR using high-resolution melting.

Infections caused by resistant microorganisms are a complex global public health challenge, and the way to combat the increase of resistance is the development of more modern and faster techniques for resistance detection. This study aimed to evaluate the transport of inactivated bacteria impregnated in a filter paper disk to detect carbapenem resistance genes by multiplex real-time PCR (qPCR) using high-resolution melting (HRM). A total of 88 isolates of 10 different species of Enterobacterales harboring well-characterized carbapenem resistance genes were evaluated. A full 10-µL loop of fresh growth of bacteria were impregnated in a filter paper disk, which was left at room temperature for 2 days in order to simulate the time spent in transportation. Bacterial inactivation was performed with 70% ethanol at 15 min. Afterwards, the DNA was extracted from the paper disks for further analysis by qPCR HRM. The time of 15 min in 70% ethanol was enough to inactivate all the isolates tested. It was possible to correctly identify the presence of the carbapenem resistance gene by HRM qPCR in 87 isolates (98.87%) that were transported in the filter paper disks. Our results indicated that it is possible to use filter paper to transport inactivated bacteria and to identify carbapenem resistance genes by qPCR HRM. This alternative tends to facilitate the access to this technology by many laboratories which do not have the qPCR equipment.

Genotyping of the Major SARS-CoV-2 Clade by Short-Amplicon High-Resolution Melting (SA-HRM) Analysis.

The genome of the SARS-CoV-2 virus, the causal agent of the COVID-19 pandemic, has diverged due to multiple mutations since its emergence as a human pathogen in December 2019. Some mutations have defined several SARS-CoV-2 clades that seem to behave differently in terms of regional distribution and other biological features. Next-generation sequencing (NGS) approaches are used to classify the sequence variants in viruses from individual human patients. However, the cost and relative scarcity of NGS equipment and expertise in developing countries prevent studies aimed to associate specific clades and variants to clinical features and outcomes in such territories. As of March 2021, the GR clade and its derivatives, including the B.1.1.7 and B.1.1.28 variants, predominate worldwide. We implemented the post-PCR small-amplicon high-resolution melting analysis to genotype SARS-CoV-2 viruses isolated from the saliva of individual patients. This procedure was able to clearly distinguish two groups of samples of SARS-CoV-2-positive samples predicted, according to their melting profiles, to contain GR and non-GR viruses. This grouping of the samples was validated by means of amplification-refractory mutation system (ARMS) assay as well as Sanger sequencing.

Epigenetic regulation of folate receptor-α (FOLR1) in human placenta of preterm newborns.

INTRODUCTION: Folates are essential nutrients for fetal development and pregnancy outcomes; they are transported to the fetus during gestation through specific folate transporters located in the placenta. In preterm newborns, we previously showed a lower placental mRNA expression of FOLR1 along with higher folate and lower vitamin B12 cord blood levels. Thereby we aimed to explore FOLR1 methylation in placentas of preterm newborns and hypothesized an increased FOLR1 methylation associated with cord blood folates and vitamin B12 concentrations. METHODS: FOLR1 methylation and mRNA were determined by methylation sensitive - high resolution melting (MS-HRM) and by real-time PCR respectively, in two placental sides of placental tissues: maternal (basal, BP) and fetal plates (chorionic, CP) of moderate preterm infants (32-36 gestational age) and term birth (37-41 gestational weeks). Folates and vitamin B12 were determined by electrochemiluminescence in umbilical cord blood samples from term and preterm newborns. RESULTS: We found that in preterm newborns, FOLR1 mRNA was lower in both plates of placenta compared with term newborns (p < 0,05) and was negatively associated with methylation of FOLR1 in CP. Preterm newborns presented higher folate and lower vitB12 concentrations in cord blood which correlated with increased placental FOLR1 methylation. DISCUSSION: In preterm newborns, placental FOLR1 expression is regulated by epigenetic mechanisms and presumably by maternal concentrations of folate and vitamin B12.